Recombinant DNA technology revolutionized the fields of biochemistry and molecular biology over the past decade. It is now possible to produce proteins, heretofore available only in trace amounts, in commercial quantities by transforming bacteria with nucleotide sequences encoding the desired protein. Efficient application of this technology to the commercial production of pharmaceuticals and other valuable substances requires that the production of the desired product be optimized.
In recombinant bacteria, for example, the transforming sequences are usually inserted into plasmids--extrachromosomal rings of double-stranded DNA. Bacteria transformed with these recombinant plasmids produce the protein encoded by transforming sequences ("product protein") in substantial yields.
The product protein is often heterologous; that is, it does not occur naturally in the host. Because the rate at which product protein is synthesized is generally limited by the quantity of messenger RNA ("mRNA") transcribed from the transforming DNA, the rate at which product protein is synthesized may be increased by increasing the number of transforming sequences per host cell. The number of such sequences is referred to herein as copy number.
In order to maximize product synthesis, bacterial host cells are transformed with many plasmids harboring the transforming sequence. Typically, the number of such plasmids ranges from 20 to 30 vectors per host cell. Copy number may also be increased by inserting multiple copies of a transforming sequence into each plasmid. See e.q., D. R. Moser and J. L. Campbell, 1983, "Characterization and Complementation of pMB1 Copy Number Mutants: Effect of RNA1 Gene Dosage on Plasmid Copy Number and Incompatibility", J. Bacteriol., 154:809-181.
However, simply increasing the quantity of transcript is not sufficient to optimize protein synthesis by transformed cells. The product protein may be toxic, to varying degrees, to the host cell. Expression of the transforming sequences at high levels also generally reduces host cell viability because such expression usurps the synthetic machinery of the host.
Moreover, recombinant bacteria often lose the transforming plasmids over time, resulting in a mixture of productive and non-productive cells. Because of the metabolic burden imposed by replication and production of the product of the transforming sequences, over time a population of transformed cells is eventually overtaken by wild-type cells, either pre-existing or resulting from segregational instability, competing with the transformed cells for available nutrients in a bioreactor.
A generally useful strategy for addressing these limitations is to grow transformed cells to a high density, while suppressing synthesis of the product protein. After the transformed cells have been grown to the desired density, a production phase is initiated by induction or derepression of transcription of the structural gene for the product protein. It is therefore necessary to retain control of the promoter for the structural gene for the product protein during the growth phase, while providing a high rate of protein transcription following induction or derepression.
While outlined here in the context of recombinant bacteria, similar considerations apply to other recombinant cells such as yeast, fungi, and higher eukaryotic cells.